Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins

DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency.

further digested for 2 h by using XhoI restriction enzyme (Thermo Scientific) to reduce the Fab fragment background in the construction of the DARPin PelB signal sequence libraries.
Subsequently, the digested libraries carried by the pEB32x vector were extracted from an agarose gel with GeneJet Gel Extraction kit (Thermo Scientific). SfiI digested anti-GFP DARPin gene was ligated into the synonymous n-region and hydrophobic region libraries in 1:3 vector/insert molar ratios by using T4 DNA Ligase (Thermo Scientific). The ligation reactions were incubated for 2 h at RT and inactivated for 5 min at 70 °C. Both ligation reactions, which contained ~300 ng of pEB32x vector DNA in 25 µl volume, were mixed with 280 µl of E. coli SS320 (MC1061 F') cells [34] and transformed into the cells with Bio-Rad Genepulser (Bio-Rad, Hercules, USA) with settings 2.5 kV, 25 µF, 200 Ω in two separate electroporation. After electroporation, the cells were recovered in 10 ml of SOC medium at 37 °C for 45 min with 100 rpm shaking. In order to determine the sizes of the two libraries, the recovered cells were diluted 10 -1 -10 -5 in SB medium, and 100 µl of the 10 -3 -10 -5 dilutions were plated on LA plates (0.5 % glucose, 25 µg/ml cm, 10 µg/ml tet). Plates were incubated at 37 °C, o/n. The rest of the cells were plated on big LA plates (0.5 % glucose, 25 µg/ml cm, 10 µg/ml tet) and incubated at 30 °C, o/n. The next day, cells were scraped off from the plates with 2 ml of SB medium (2 ml/plate). The library sizes of the DARPin N (synonymous alterations in the n-region of the PelB) and DARPin H (synonymous alterations in the hydrophobic region of the PelB) were 1.9 x 10 7 cfu and 1.3 x 10 6 cfu, respectively.

Construction of a serine/tyrosine binary library
Combined PelB signal sequence mutants of DN5 and DH4 (DN5-DH4) and DN10 and DH4 (DN10-DH4, assayed only in previous early phage display experiments, see Figure S2) were created by amplifying the pEB32x-DARPin PelB DN5 and DN10 vectors with PCR using common forward primer TH361 and mutant-specific reverse primers TH359 (for DN5-DH4) S3 and TH360 (for DN10-DH4), respectively. The vector fragments were ligated to TetR cassette (stuffer fragment of 2101 bp) with SfiI restriction enzyme and the correct double mutant sequences were verified by sequencing. SfiI vector backbones from pEB32x-DARPin vectors parental PelB, DN5, DH4 and DN10, as well as, from pEB32x-Tet double mutant vectors DN5-DH4 and DN10-DH4 were gel extracted and ligated with a combinatorial DARPin library SfiI DNA insert containing serine/tyrosine codon variation (TMY) at specified codon positions ( Figure S1). The DARPin binary library was purchased as ready linear DNA block from Eurofins Genomics (Ebersberg, Germany), amplified with PCR using added flanking primer sites and digested with SfiI. The ligated library DNA samples were transformed to E.
coli XL-1 Blue cells with electroporation each yielding >10 000 cfu transformants. All PelB mutant libraries contained < 0.7% vector background colonies, which were calculated from the transformation plates of vector control ligations (no insert) that were prepared in parallel to the library samples.
As DsbA sequence cannot contain a compatible SfiI site for receiving the DARPin binary library SfiI DNA fragment, DsbA-DARPin library was constructed with Gibson assembly. The vector fragment containing DsbA was PCR amplified with primers TH363 and TH364 using pEB32x-DsbA-DARPin anti-GFP vector as the template. The DARPin binary library was amplified with primers TH362 and JLe01as using peB32x-DARPin library (parental PelB) as the template. Gibson reaction was performed according to Gibson 2009 paper [37]. The resulting pEB32x-DsbA-DARPin library was further purified by PCR amplification with primers WO375 and PAKrev, XbaI and HindIII digestion and gel extraction of the correct size fragment covering DsbA-DARPin-p3-CT gene. The insert was ligated to XbaI and HindIII digested pEB32x vector fragment and electroporated to E. coli XL-1 Blue cells yielding 1.6 x10 4 cfu transformants with 3% vector background colonies that were calculated as above. The library cloning strategy was confirmed by sequencing ten DN10-DH4 (same insert in all PelB libraries) and ten DsbA clones. In both library samples 7/10 clone sequences were according to the library design and 3/10 frameshift clones.

Cloning of pAK400-TrxA and -DARPin periplasmic export constructs
Soluble DARPin anti-GFP periplasmic expression vectors (WT, DN5 A8V, DN5 V3 and DN5-DH4) were constructed by inserting XbaI-PstI fragments from pEB32x-DARPin (contain signal sequence and DARPin gene) into pAK400-DARPin PstI-XbaI backbone and verified by S4 sequencing. For the TrxA export reporter constructs, TrxA was amplified with primers TH393 and TH394 (Table S1) from the genome of E. coli XL-1 Blue cells with Phusion DNA polymerase. To this end, a colony of cells was resuspended in 50 µl H2O, boiled for 5 min at 98 °C and, 1 µl of thermally lysed cells was added to a 50 µl PCR reaction as template. PCR was cycled for a total of 40 cycles (init. den. at 98 °C for 30 s, den. at 69 °C for 10 s, ext. at 72 °C 30 s and final ext. at 72 °C for 5 min) and the product purified with DNA Clean & Concentrator-5 Kit (Zymo Research, USA). TrxA was cloned with flanking SfiI sites to pAK400 and verified by DNA sequencing. pAK400-TrxA PelB sequence variants (WT, DN5 A8V, DN5 V8A, DN5 V3 and DN5-DH4) were prepared by replacing DARPin gene in pAK400 vectors by TrxA gene with SfiI. The pAK400-DARPin DsbA control used for the western blotting experiment was cloned with Gibson assembly as described above using primers TH419, TH420, TH421 and TH422. PhoAWT-TrxA, PhoAGTG-TrxA and TrxA without signal sequence were cloned by amplifying TrxA gene form pAK400-TrxA PelBWT with forward primers TH436, TH437 and TH438 and reverse primer pAKrev2 and, cloning the PCR products to pAK400 vector as XbaI-HindIII fragment.

LC-ESI-MS
The purified periplasmic DARPin samples were diluted to 0.1 µg/µl concentration with ultrapure water and 0.2 µg protein was used for LC-ESI-MS. The LC-ESI-MS analyses were performed on a nanoflow HPLC system (Easy-nLC1000, Thermo Fisher Scientific) coupled to the Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a nano-electrospray ionization source. The samples were first loaded into a trapping column (2 cm, 100 µm i.d.) and then separated online in an analytical column (15 cm, 75 µm i.d.). The both columns were in-house packed with ReproSil-Gold 300 C4 3 µm column packing material (Dr. Maisch Gmbh, Germany). The mobile phases consisted of a solvent A (water with 0.1% formic acid) and a solvent B (acetonitrile/water (80:20 (v/v)) with 0.1% formic acid). The online desalting was carried out by loading the samples into the precolumn with 22 µl volume of the solvent A. A 20 min chromatographic method was used to elute proteins: from 12% to 65% of the solvent B in 14 min, following from 65% to 100% of the solvent B in 3 min, and finally 3 min wash at 100% solvent B. A flow rate was 400 nl/min.

S5
MS data was acquired automatically by using Thermo Xcalibur 4.4 software (Thermo Fisher Scientific). A scan range from m/z 420 to m/z 2000 at 7500 orbitrap resolution was used in the MS method. A performance of the instrumentation was checked by analyzing a protein standard mixture (Pierce intact protein standard, Thermo Fisher Scientific) before the sample analyses.